Thalidomide was originally used as a sedative to treat morning sickness. However, thalidomide administration during pregnancy resulted in congenital defects in thousands of human fetuses. Many tissues and organs like eyes and heart, for example, are affected by thalidomide during embryonic development. Additionally, variable limb truncations such as amelia (absence of limbs) and phocomelia (proximal limb truncations) are frequent. Thalidomide is now used to treat leprosy and multiple myeloma. Additionally, thalidomide has been used in a treatment of a variety of other conditions including, but not limited to, chronic graft versus host disease, rheumatoid arthritis, sarcoidosis, inflammatory skin diseases and inflammatory bowel disease.
Thalidomide is a racemic chemical compound that may be described chemically as 2-(2,6-dioxo-3-piperidyl)isoindole-1,3-dione or α-(N-phthalimido)glutarimide. The empirical formula for thalidomide is C13H10N2O4, and the gram molecular weight is 258.2. The CAS number of thalidomide is 50-35-1.
Lenalidomide, a thalidomide analogue, is an immunomodulatory agent with anti-angiogenic properties. The chemical name is 3-(4-amino-1-oxo 1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione. The empirical formula for lenalidomide is C13H13N3O3, and the gram molecular weight is 259.3.
The teratogenic effect of thalidomide is species-specific. Thalidomide exposure to certain non-human primates in-utero induces limb malformations identical to those seen in humans. Certain standard model organisms like mice and rats are thalidomide-resistant, while New Zealand White rabbits and chickens show thalidomide-induced embryopathies. In humans and rabbits, the result of thalidomide treatment is primarily phocomelia, whereas in chicken limbs both proximal and distal structures are affected. In extreme cases, all three organisms display amelia.
Thalidomide induces oxidative stress through the formation of free radical-initiated reactive oxygen species (ROS) in limb bud cells and embryos of thalidomide-sensitive rabbits but not in those of thalidomide-resistant mice or rats. The cellular response to ROS production primarily consists of removal of free radicals through the reduced glutathione (GSH)-dependent detoxification pathways. GSH is oxidized to glutathione disulfide in the detoxification processes, which leads to a shift in the intracellular redox potential and results in a more oxidative environment. Dramatic oxidative intracellular redox potentials can modulate signaling and gene expression thereby inducing apoptosis. The overall embryonic redox potential and especially that of limb buds is much more oxidative in thalidomide-sensitive rabbits than in thalidomide-resistant rats. Furthermore, rat limb buds possess higher GSH stores to buffer redox potentials altered by ROS than do rabbit limb buds (1).
The soluble Wnt inhibitor Dickkopf1 (Dkk1) and canonical Wnt/β-catenin signaling are involved in limb morphogenesis. Dkk1 is known to promote programmed cell death (PCD) in the developing limb and Dkk1 expression is induced by Bmp4 and Bmp5. All bone morphogenetic proteins (Bmps) with important functions during limb development (Bmp2, -4, -5 and -7) signal through the same Bmp type I receptor (BmpR-IA) in the limb mesenchyme, and Bmp2 and Bmp7 may also regulate Dkk1 expression.
Thalidomide induces limb and eye defects in the chicken embryo at an EC50 of 50 μg/kg egg weight and apoptosis in primary human embryonic fibroblasts (HEFs) at an EC50 of 8.9 μM. Using these model systems, the present inventors demonstrate by semi-quantitative RT-PCR and whole-mount in situ hybridization that thalidomide-induced oxidative stress enhances signaling through Bmps. This leads to up-regulation of the Bmp target gene and Wnt antagonist Dickkopf1 (Dkk1) with subsequent inhibition of canonical Wnt/β-catenin signaling and increased cell death. Thalidomide-induced cell death is dramatically reduced in HEFs and in embryonic limb buds by the use of inhibitors against Bmps, Dkk1 and Gsk3β, a β-catenin antagonist acting downstream of Dkk1 in the Wnt pathway. Additionally, blocking of Dkk1 or Gsk3β dramatically counteracts thalidomide-induced limb truncations and microphthalmia. Thus, perturbing Bmp/Dkk1/Wnt signaling is central to the teratogenic effects of thalidomide.
In various embodiments, the present invention is a pharmaceutical composition comprising an anti-neoplastic agent and an anti-teratogenic agent. In further embodiments, the present invention is a pharmaceutical composition for blocking the teratogenicity of an anti-neoplastic agent comprising an agent that activates the Wnt signaling pathway downstream of the Wnt ligand-receptor interaction.
In addition to the aforementioned compositions, the present invention embodies a method of inhibiting the teratogenicity of an anti-neoplastic agent comprising administering a pharmaceutical composition comprising an agent that activates the Wnt signaling pathway downstream of the Wnt ligand-receptor interaction.
In a particular embodiment, a method of assessing the teratogenicity of a compound is disclosed. Such a method comprises: administering the compound to a cell and observing whether there is activation of the Wnt signaling pathway downstream of the Wnt ligand-receptor interaction in comparison to an untreated cell.
In yet another embodiment, there is disclosed a method of treating a mammalian subject comprising administering to said mammalian subject an effective amount of a pharmaceutical composition comprising an anti-neoplastic agent and an anti-teratogenic agent.
The included drawings and examples illustrate and exemplify the embodiments of the invention as further described herein.